All parenteral drugs are potentially hazardous in that during any stage in their production they can become contaminated. Rigid control by final testing for the presence of bacteria has been in practice for many years and standardized sterility tests such as those described in the U.S. Pharmacopoeia have proven to be adequate and effective safeguards. The problem of pyrogens, however, while controllable, is much more difficult and testing procedures are tedious, expensive and not always reproducible. As aforesaid, pyrogens are fever-causing substances, generally lipopolysaccharides which are liberated as endotoxins by gram-negative bacteria. During the processing of a drug all bacterial cells may be removed and the drug may test "sterile", yet pyrogens may still be present which could cause serious reactions when injected. For this reason all parenteral drugs must be tested for pyrogens by a procedure similar to that described in the U.S. Pharmacopoeia wherein a group of rabbits is injected intravenously with a specified amount of the drug and the resultant average temperature rise, if any, over a period of three hours is observed. Because of the obvious variation encountered in a biological test such as this, steps must be taken all along the testing procedure to ensure that conditions are as uniform and standardized as is possible. This demands a special laboratory where animals can be maintained in a basal state and an adequate supply of animals such that there is an ample recovery period between tests.
Despite the difficulties in maintaining uniform conditions, the test has proven extremely useful and is relied upon on a worldwide basis to keep pyrogenic drug reactions to a minimum. Yet a simplified, in vitro type of procedure could reduce enormously the costs of time and materials for conducting this important test. Furthermore, there are certain types of drugs, e.g. short lived radio-labeled diagnostics, cisternographic agents and certain classes of hypnotics that by their nature are precluded from the rabbit fever pyrogen test. These materials must now be used at the risk of not being pyrogen-free.
The necessity for testing an aqueous liquid containing dissolved or dispersed therein a drug such as codeine or an antibiotic, or other substance which has a medicinal function also extends to solutions or dispersions of other substances which are parenterally-administered and which are to be regarded herein and in the claims as drugs in a broad sense such as solutions of sodium chloride or of glucose that are intravenously administered or a solution or dispersion of any of various nutrients used in intravenous feeding. The necessity of testing for the possible presence of pyrogens also extends to bottles and other containers for parenterally-administered liquids and likewise to tubing, injection needles and other instrumentalities used in the parenteral administration of drugs or used in connection with transfusions or infusions. Testing for contamination by pyrogens also has application to water and saline solutions intended for use in parenterally-administered solutions as well as for other purposes. For example, the presence of pyrogen in potable water has been shown to have a definite relationship to bacteria population. Testing for the presence of pyrogens also has other applications such as the determination of their possible presence in certain of the body fluids of mammals.
An indication that an in vitro test might be available appeared several years ago when it was discovered that the circulating cell, the amebocyte, in the blood of the horseshoe crab (Limulus polyphemus) contains a group of proteins that react with low levels of endotoxins. Evidence was obtained to show that one of three proteins, probably a proteolytic enzyme, is activated in some as yet unknown manner by endotoxin after which it causes another protein to coagulate or gel. The protein mixture (lysate) may be isolated from the amebocytes and used as a reagent to detect the presence of endotoxin by noting the formation of a gel after incubating lysate and endotoxin for an hour or more.
In the test procedure most generally employed heretofore the lysate is prepared by washing the amebocyte cells contained in the blood of the horseshoe crab so as to remove the components of the blood other than the amebocyte cells. This usually is accomplished using water containing sodium chloride in order to minimize premature deterioration of the walls of the cells. Preferably, the washing solution also contains calcium chloride. The washing also is accomplished in the presence of N-ethylmaleimide which has been found to be effective in preventing the aggregation of amebocyte cells so that they may be separated more readily from the aqueous wash water by centrifugation. Two such washings followed by centrifugation are normally sufficient. The washed cells thereafter are lysed using 2 volumes of water for each volume of the washed cells. After the lysing has been completed, as by agitating the suspended cells, the protein content of the cells becomes dispersed in the water, which becomes the "lysate" after the separation of the cell debris by centrifugation. Other lysing techniques also are known.
In carrying out the gel test hereinabove mentioned, it has been the usual practice heretofore to add 0.1 ml of the aqueous solution as prepared for parenteral administration to 0.1 ml of the lysate such as that produced as previously described. After incubation of the mixture at 37.degree. C. for 1 hour the mixture is examined for gel formation. If gel is formed, this is an indication of the presence of an endotoxin due to coagulation of the protein content in the lysate. The usual manner of evaluation as to whether or not a gel has formed is by the ability of the mixture to remain in the tube in which the test is carried out when the tube is inverted. Usually the test also is run with a known level of endotoxin as a comparative standard. However, because of variation from lot to lot of lysate and variation in the nature of different endotoxins, gel formation is not always uniform and evaluation often becomes a subjective determination. Further, for very low levels of endotoxin, the incubation period is prolonged and occasionally a thickening of the mixture is observed or a formation of granules without the formation of a solid clot thereby rendering the test even more difficult to evaluate.